A candidate with broad reach
A glycan called R3 was associated with stronger immune-cell activity against MCF-7 cells in laboratory tests. The researchers also reported strong interaction with Siglec-5, Siglec-7, Siglec-9, Siglec-10 and Siglec-11. In the functional assays, R3 was linked to higher macrophage phagocytosis, the measured uptake of target cells, and higher cytotoxicity by natural-killer cells and macrophages.
The study set out to find a common small-molecule inhibitor that could target multiple Siglecs involved in immune-checkpoint suppression. To search for one, investigators chemoenzymatically synthesized more than 60 glycans and assembled them on a glycan microarray, a testing surface used to compare binding to different sugar structures. The work then compared candidates labeled R1 through R5.
On the Siglec-10 array, unsialylated R1 had no detected binding. R3 and R5, which carried terminal Neu5Gc, showed higher affinity than R2 and R4, which carried terminal Neu5Ac, and the reported pattern favored the 2,3 linkage over the 2,6 linkage. R3's apparent dissociation constant was reported as 0.050 ± 0.011 μM.
Flow-cytometry inhibition tests gave a more specific comparison. R1 showed no apparent interference at 200 μM. R3 showed significant blocking at 300 μM, while R5 was compared at 1 mM. In a broader array, R3 showed strong interaction with Siglec-5, Siglec-7, Siglec-9, Siglec-10 and Siglec-11. The study presented R3 as a candidate for a common, multi-Siglec checkpoint blocker.
The researchers also profiled the N-glycans on MCF-7 cells, meaning the sugar structures measured on their proteins. About 70% of detected compositions were high mannose, fewer than 10% contained sialic acid, and more than 97% of the detected sialic acids were Neu5Ac, with traces of Neu5Gc. In a separate test, Siglec-10 still bound CD24-knockout MCF-7 cells despite absent CD24 expression. This residual binding means the interaction observed in the assay could not be attributed solely to CD24.
Changes in cell assays
R3 was then tested in macrophage assays against MCF-7 cells. After two hours, reported average phagocytosis was higher than with the PBS control across all four macrophage states. The reported enhancements were 56.2% for IL-4-induced M2-like macrophages, 137.9% for IL-10-induced M2-like macrophages, 82.2% for M0-like macrophages and 22.7% for M1-like macrophages. These assays used three donors and normalized results to the highest value in each experiment, so the percentages describe relative enhancements rather than absolute shares of cells engulfed.
R3 was also associated with stronger killing activity. After six hours, NK-cell cytotoxicity was 54.4% higher and macrophage-mediated cytotoxicity was at least 46.3% higher. The figure was based on three donor biological replicates and used paired, two-tailed t tests on original raw data. It reported significance markers at p below 0.05 and p below 0.01.
Finally, the team tested antibody-dependent cellular phagocytosis, or ADCP, using R3 with trastuzumab. Among M2-like macrophages, R3 alone at 1 mM was associated with a 100.4% enhancement, trastuzumab alone at 0.02 μg/mL with a 145.3% enhancement, and the combination with a 258.9% enhancement. For M0-like macrophages, the corresponding figures were 70.0%, 80.3% and 198.8%. The ADCP figure used four donors and normalized results to the highest value in each experiment, with the combined treatment producing the largest reported normalized result in both macrophage states.
What the results leave open
The authors interpret R3 as a common inhibitor capable of engaging several glyco-immune checkpoints and potentially enhancing macrophage phagocytosis, NK-cell cytotoxicity and antibody effector functions. The functional results came from assays with three donor replicates, while ADCP used four, and the reported enhancements were normalized rather than absolute rates. Structural studies of R3 binding to Siglecs are underway, so the molecular binding mode has not yet been established. Because the evidence described here comes from glycan arrays and cell assays, it does not establish how R3 would perform in patients.
Paper data and sources
Original title: A Common Small-Molecule Inhibitor Targeting Multiple Siglecs Associated with Immune Checkpoint Suppression of Cytotoxicity and Phagocytosis
Authors: Chung C, Li R, Cheng L et al.
Journal/Repository: Journal of the American Chemical Society
Status: Peer-reviewed
First online: 2026-10-19
DOI: Not available
Original paper · Full text